Viral Synthetic Genomics to Engineer Large dsDNA Viruses

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Viruses with large double-stranded DNA (dsDNA) genomes are a diverse group, which includes members that can infect a wide range of different hosts. Some of these viruses are important pathogens of humans and animals. Improving our ability to manipulate their genomes rapidly and accurately will increase our understanding of how these viruses replicate and cause disease and the likelihood of developing vaccines. Utilizing synthetic genomics tools developed by scientists at the J. Craig Venter Institute during the construction of the synthetic Mycoplasma genome, we have developed a novel application that allows us to make genome-wide changes in the virus more rapidly than previous methods. The overall strategy uses transformation-associated recombination (TAR) in Saccharomyces cerevisiae to clone overlapping fragments of the virus genome, which can then be manipulated by many techniques in E. coli, yeast or in vitro, and reassembled into complete genomes.

We have projects developing synthetic genomics approaches and reagents for several human herpesviruses, herpes simplex virus type 1, human cytomegalovirus and Epstein-Barr virus. Herpesviruses have large genomes, with relatively high GC content, and characteristic regions of repeated sequence and because of these features, is difficult to manipulate with current methods. Synthetic genomics approaches reduce the time needed to make complex, multi-loci mutations in these viruses substantially. We are also using these techniques to bring genetic tools to African swine fever virus with the goal of developing a vaccine to control outbreaks of the disease, that can have significant economic impact, especially in Sub-Saharan Africa.

These approaches also improve our ability to develop and utilize dsDNA viruses as diagnostics and therapeutics for a range of diseases. The capacity to rapidly design, build and test engineered large dsDNA viruses could lead to the development of better vectors for gene therapy or DNA vaccines, or improved oncolytic viruses.

Funding for this project provided by US National Institutes of Health R21AI109418 (S.V.), NIH R21AI109338 (P.J.D and S.V.), US National Institutes of Health 1R41AI106090-01A1, and International Development Research Center Grant # 108514-001.